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cytokines in classical swine fever virus subunit
gp55/E2-DNA vaccination
Daniel Wienhold, Elisenda Armengol, Annette Marquardt, Christian
Marquardt, Heiner Voigt, Mathias Büttner, Armin Saalmüller, Eberhard Pfaff
To cite this version:
DOI: 10.1051/vetres:2005019
Original article
Immunomodulatory effect of plasmids co-expressing
cytokines in classical swine fever virus subunit
gp55/E2-DNA vaccination
Daniel W
IENHOLDa, Elisenda A
RMENGOLa, Annette M
ARQUARDTa,
Christian M
ARQUARDTa, Heiner V
OIGTa, Mathias B
ÜTTNERa,
Armin S
AALMÜLLERb, Eberhard P
FAFFa*
a Friedrich Loeffler Institute, Institute of Immunology, Paul-Ehrlich-Strasse 28, 72076 Tübingen,
Federal Republic of Germany
b Clinical Immunology, University of Veterinary Medicine Vienna, Veterinärplatz 1,
1210 Wien, Austria
(Received 28 April 2004; accepted 2 December 2004)
Abstract – The aim of this study was to determine the immunomodulatory effects of IL-12, IL-18
and CD154 (CD40 ligand, CD40L) in DNA-vaccination against the classical swine fever virus. Four recombinant plasmids were constructed including the CSFV coding region for the glycoprotein gp55/E2 alone or together with porcine IL-12, IL-18 or CD154 genes. Five groups of four pigs each were immunized intramuscularly (i.m.) three times with the respective constructs. The control group was inoculated with empty plasmid DNA. Eighteen days after the final immunization, the pigs were challenged with a lethal dose of CSFV strain Eystrup and monitored for a further 16 days. This study showed that co-delivery of IL-18 and CD154 induced an earlier appearance of serum antibodies, reduced B-cell deficiency after infection and protected pigs against a lethal CSFV infection. In contrast, co-delivery of IL-12 led to a reduced titer of neutralizing antibodies and protection against a lethal CSFV challenge in comparison to the other pigs and to pigs that were immunized with a gp55/E2 plasmid alone.
DNA vaccination / classical swine fever virus
1. INTRODUCTION
Classical swine fever virus (CSFV) is a member of the genus Pestivirus within the Flaviviridae and causes a highly infectious and fatal disease in pigs. Infection of swine with classical swine fever virus is charac-terized by fever, haemorrhages and mortality [9]. In several European countries,
out-breaks of CSFV occur periodically in domestic pigs and are always combined with high economic losses [27]. Although highly efficacious, the use of conventional, attenuated live virus CSFV-vaccines do not allow a distinction between naturally infected and vaccinated pigs, therefore the European Union (EU) currently pursues a non-vacci-nation policy including the stamping-out of
* Corresponding author: eberhard.pfaff@tue.bfav.de
infected herds. Marker vaccines based on recombinant glycoprotein E2 allow discrimi-nation between vaccinated and infected ani-mals. Several groups working on CSFV vaccines have shown that especially the E2-protein is able to induce a specific and pro-tective immune response against CSFV after vaccination with an E2-expressing recombinant virus [17, 22, 32], by immuni-zation with baculovirus expressed E2 pro-tein [5, 44, 45]. These experiments have demonstrated that glycoprotein E2 is the most immunogenic and protective protein of CSFV inducing e.g. a high neutralizing antibody titer [1, 45].
Recombinant baculovirus expressed E2-glycoprotein vaccines have been on the market for several years and are somewhat effective, however they are not able to inhibit virus spread from infected animals [12]. Therefore the efficacy of the vaccine is incomplete.
Another approach for more effective vaccines against CSFV infection is based on DNA-vaccines [1]. Vaccination with E2-DNA allows the induction of a long term immune response and as mentioned above the serological differentiation between vacci-nated and infected animals [44]. Neverthe-less the immunogenicity of the DNA-constructs is sometimes low and the immu-nostimulatory capacity might be improved by other components like cytokine genes or CpG-motifs [4, 20, 40].
For other species, it has been previously described that co-administration of cytokine genes with DNA-vaccines increases the immune response and modifies the immune response towards a Th1- or Th2-type reac-tion [4, 14, 20, 38]. In various infecreac-tions the Th1 or Th2 differentiation of helper T lym-phocytes is pivotal to lead to a protective immune response.
In order to enhance the immunostimula-tory effect of an E2-DNA vaccine and to direct the E2-specific immune response towards a protective immunity, we used 3 different genes of proteins involved in the onset and control of an immune response:
genes of two cytokines, IL-12, IL-18 and the sequence of a regulatory cell surface molecule; CD154 or CD40L. IL-12 is a het-erodimeric molecule composed of the cova-lently linked subunits p35 and p40. It is secreted by peripheral B- and T-cells after induction through bacteria, bacterial prod-ucts, and parasites [10] as well as by den-dritic cells [19]. IL-12 induces a strong IFN-γ expression in NK cells [24, 49] and promotes a Th1 response under most exper-imental conditions [4, 38]. Other cytokines such as IL-18, a monomeric molecule, which is expressed as a 24 kDa precursor protein [28] and after being processed by the IL-1-β converting enzyme, are able to stimulate both the Th1 and the Th2 immune response depending on the surrounding cytokine milieu [29]. Finally, CD154 (CD 40L) as a type 2 transmembrane protein has been reported to be expressed on activated T- and B-cells as well as on natural killer-and dendritic cells [8, 30]. The CD40L-CD40 interaction influences and sustains co-stimulatory signaling in APC which is essential for T-cell dependent antibody response [3, 16]. The CD154 activation of immune cell populations is mainly classi-fied as the Th2-response, depending on the immunological microenvironment.
For a better understanding of the porcine immune response against CSFV, the effect of co-delivery of the porcine immunomod-ulating genes on the efficacy of a CSFV E2-DNA vaccine has been tested in domestic swine in a stringent CSFV challenge trial.
2. MATERIALS AND METHODS 2.1. Cloning and DNA preparation
Classical swine fever virus glycoprotein gp55/E2 (U45478) was isolated and cloned (HindIII/NotI) into the eucaryotic plasmid expression vector pcDNA4HisMax using standard protocols.
NotI/XhoI/NruI/StuI) were inserted into the pcDNA3 plasmid KpnI/StuI. The genes encoding porcine IL-18 (U68701) and CD154 [48] were first isolated using prim-ers including KpnI/NotI restriction sites and subcloned in the modified pcDNA3 plasmid. The IL-12 p35 and p40 subunit genes were isolated as described previously [15]. To allow the expression of the het-erodimeric IL-12 protein, the p40 subunit was linked to an IRES sequence from the pIRES1neo plasmid (Clontech, Palo Alto, USA) followed by the p35 subunit and was also subcloned (BamHI/SalI ligated to XhoI) in the modified pcDNA3 plasmid. A second separate transcription unit including the CMV promotor and the cytokine genes was isolated from the modified pcDNA3 plasmid after a NruI digestion. Completing the second transcription units were ligated to the cleaved (PmlI) pcDNA4-E2 plasmid. The gene sequences were verified by the dideoxy nucleotide chain termination method using the BigDye Terminator Cycle Sequencing v2.0 Kit (Applied Bio-systems, Foster, USA) and were analyzed by the University of Wisconsin Genetics Computer Group (UWGCG) Software (Accelrys, Munich, Germany) [11]. The plasmid DNA was purified by caesium chloride centrifugation for vaccination in pigs.
2.2. Transfection and expression
To check the transient expression of por-cine CD154 and the classical swine fever virus glycoprotein gp55/E2, 5 × 105 porcine MAX-cells [31] were seeded and incubated for 24 h. The MAX-cells were transfected using 1 µg DNA and Effecten® (QIAGEN,
Hilden, Germany) according to the manu-facturer’s protocol. Twenty-four hours after transfection, the cells were analyzed by immunofluorescence for CD154 and gp55/ E2 expression using specific monoclonal antibodies. For the immunofluorescence staining, transfected cell monolayers were washed once with PBS and fixed with 100% EtOH for 15 min at room temperature for
the detection of CD154 expression or 20 min with 3% formaldehyde including 10% Tri-ton X for the analysis of the E2 expression. After fixation, the cells were washed three times with PBS and incubated either with a mouse monoclonal anti-human CD154 (5C8, ATCC, Manassas, VA, USA) or with the mouse monoclonal anti-E2 antibody a18 [46] for 1 h at room temperature. After incubation, the cells were washed three times with PBS and stained with propidium iodide. The binding of the CD154 or E2 spe-cific antibodies was visualized with fluo-rescein-isothiocyanate (FITC)-conjugated anti-mouse IgG antibodies (Dianova, Ham-burg, Germany).
For the detection of the expressed por-cine IL-12 and IL-18, the supernatant of transiently transfected MAX-cells was col-lected 24 h after transfection. Since there was a lack of specific monoclonal antibod-ies against porcine IL-12 and IL-18, their production was analyzed using their bio-logical activity to enhance the cytolytic activity of natural killer (NK) cells. Assays for the detection of the activity of natural killer cells were performed as described [7, 36]. In brief, K562 tumor cells were labeled with Na51CrO4 (Amersham, Braunschweig,
Germany) for 90 min (100µCi/106 cells), washed and incubated for 16 h along with freshly isolated effectors (PBMC) at an effector-to-target cell ratio of 100:1. The per-centage of specific cytolytic activity was cal-culated as described previously [35].
2.3. Vaccination experiment and challenge
Five groups of four 6 week-old conven-tional pigs were each immunized intramus-cularly (i.m.) with 1000µg of the respective plasmids. All pigs received two boost immunizations (1000µg) on day 22 and 44 post vaccination (p.v.). 18 days after the last immunization, the pigs were challenged with a lethal dose of 105 TCID
50 of CSFV
clinical signs, the pigs were controlled daily by measuring rectal body temperature. Pos-sible viraemia and virus excretion was tested on days 4, 7, 10 and 14 after challenge by collecting blood samples and nasal swabs as well as organ samples on the day of slaughter.
2.4. Antibody response
All sera were tested in an ELISA for E2-specific antibodies (Chekit®, Bommeli Diagnostics, Bern, Switzerland) and in a neutralization peroxidase-linked assay (NPLA) [41] for the titer of CSFV-specific neutralizing antibodies. NPLA titers are expressed as the reciprocal of the serum dilution that neutralizes 100 TCID50 of
CSFV strain Glentorf in 50% of the repli-cate culture.
2.5. Virus isolation
The collected blood, organ samples and nasal swabs of the pigs were quantitatively tested for the presence of viable virus (TCID50) using an immuno-peroxidase monolayer assay (IPMA) as described [47]. Virus titers were calculated as log10 TCID50/mL.
2.6. Isolation of PBMC and FACS analyses
Peripheral blood mononuclear cells (PBMC) were separated from venous blood using density gradient centrifugation with Ficoll-Hypaque (PAA, Linz, Austria) [34]. Staining of PBMC for flow cytometric analyses was performed by incubation with a monoclonal antibody against human CD21 showing reactivity with the porcine CD21 (B-ly4, IgG1, BD Pharmingen, San Diego, CA, USA) followed by incubation with an isotype-specific PE-conjugated goat-anti mouse IgG1 antibody (Southern Biotech, Birmingham, AL, USA). All incu-bation periods were done on ice for 20 min. After each incubation step, the cells were washed twice with FACS buffer (PBS
with-out Ca2+ and Mg2+, 2% FCS). Labeled cells
were resuspended in FACS buffers and ana-lyzed in a FACStarplus (BD Biosciences, Mountain View, CA, USA) as described [36]. The percentages of CD21-positive B lymphocytes were calculated using Win-MDI or Cell Quest® software (BD
Bio-sciences).
3. RESULTS
3.1. Cloning and in vitro expression experiments
The gene encoding the full length E2/ gp55 protein from CSFV was isolated and cloned into the expression vector pcDNA4HisMax (Fig. 1A). The isolated and subcloned genes for IL-12, IL-18 and CD154 were additionally inserted into the pcDNA4HisMAX-E2 plasmid using a sep-arate transcription unit (Fig. 1B). The expression of the encoded E2 glycoprotein and the porcine CD40L was confirmed by immunofluorescence staining, after trans-fection into MAX cells, using a monoclonal anti-E2 antibody for the E2 glycoprotein (Fig. 2A.) and a cross reacting monoclonal anti-human CD40L antibody for the por-cine CD40L (Fig. 2B). All tested constructs showed high expression of the E2 glycopro-tein after transfection into MAX cells. Even high amounts of CD40L were expressed after transfection with pcDNA4-E2-CD40L into MAX cells.
The increase of the NK activity by the supernatant of pcDNA4HisMAX-E2-IL-12 transfected MAX cells is comparable with the effect of the adenoviral
recom-binant porcine IL-12 supernatant which served in all assays as a positive control.
Comparable to the effect of IL-12, we also detected an enhancement of NK activ-ity by adding the supernatant of pcDNA4-HisMAX-E2-IL-18 transfected MAX cells to the NK cells. As mentioned above, the pcDNA4HisMAX-E2 plasmid served as the control. With these results, we were able to demonstrate in a cell based assay the bio-logical activity of the pcDNA4-E2-IL-12 and pcDNA4-E2-IL-18 plasmids, which were thereafter used in the in vivo immuni-zation experiments.
3.2. CSFV antibody response in DNA vaccinated pigs
Previous reports about vaccination trials against CSFV present a correlation between humoral immune response and protection. Therefore, we decided to monitor the suc-cess of the DNA-immunization with the respective plasmids and the influence of the addition of the immuno-regulatory mole-cules IL-12, IL-18, and CD154 in a first step by the development of CSFV-specific neu-tralizing antibodies after vaccination. All pigs received a single immunization and two boost immunizations in a 22 day-inter-val. The onset and the magnitude of the humoral immune response illustrated by the appearance and the titer of CSFV-spe-cific neutralizing antibodies is presented in Table I. The animals in group No.1 were immunized with naked pcDNA4HisMAX plasmid DNA and served as positive con-trols for the virus challenge. As expected, none of the animals showed a detectable CSFV-E2-specific antibody titer after vac-cination and the time after the challenge infection was too short for the development of virus-specific antibodies. All control ani-mals had to be slaughtered 6–7 days after the challenge infection. Group 2 animals were immunized with the pcDNA4HisMAX-E2 plasmid. The animals showed heteroge-neous antibody titers after vaccination. In one swine, the antibody titer was very low, Figure 1. The E2 sequence from CSFV strain
whereas three out of four pigs developed CSFV-specific neutralizing antibodies with a reciprocal serum dilution of 60–1600 for neutralizing antibodies before challenge
infec-tion against 100 TCID50 of CSFV strain Glentorf in the neutralizing peroxidase-linked antibody assay (NPLA). All pigs showed an increase of antibody titers after Figure 2. Immunofluorescence analysis. (A) The porcine MAX cell line was transfected with the
challenge infection. Two pigs developed up to > 1600 antibody titers ten days post-infection. The animal with the low titer of neutralizing antibodies prior to the infec-tion kept the low antibody titer and showed only an increase of a reciprocal NPLA-titer of 256 on 14 dpi. One other animal (animal No. 4) died under sample collection on day 7 post infection.
The antibody response of group 3, which included pigs receiving the
pcDNA4HisMAX-E2-IL-12 plasmid, was poor. Only one of four pigs had a detectable NPLA-titer before challenge infection and only two pigs showed a detectable antibody response after challenge. Two pigs, which were not protected by the vaccination had to be euth-anized 6–7 days after challenge infection without measurable antibody titers. In con-trast, in group 4, treated with the pcDNA4-HisMax-E2-IL-18 plasmid (Tab. I, group 4) three out of four animals developed a Figure 3. Dose-depended enhancement of NK activity by IL-12 and IL-18. PBMC were incubated
detectable titer of neutralizing antibodies (day 50, titer 20–40) 6 days after the second boost immunization. One individual showed a time-delayed antibody response with an equivalent titer one week later. Interest-ingly, after the first immunization with the plasmid, the behavior of the fourth animal on this group showed a neutralizing antibody titer of 1/60, which might be interpreted as a very effective immunization. As expected, after the challenge infection, the antibody titers increased in all individuals of the E2-IL-18 group very soon, so that 7 days after the challenge infection in nearly all animals,
neutralizing antibody titers > 1000 were obvious. A similar behavior was seen in group 5. These swine were immunized with the pcDNA4HisMax-E2-CD40L plasmid. Three of four pigs showed a significant titer 6 days after the second boost immunization and the fourth one week later. But compared to the former group, the titer was slightly diminished. After challenge infection, all members of this group developed high titers of neutralizing antibodies, but compared to the E2-IL-18 group, they were time-delayed. Whereas most of the animals of group 4 showed a titer of about 1/1000, 7 days after Table I. NPLA (neutralizing peroxidase-linked antibody assay) results are expressed as the
recip-rocal of the serum dilution neutralizing 100 TCID50 of CSFV strain Glentorf in 50% of the replicate culture. The time points of serum sampling after challenge infection (day 61) are indicated in bold.
Plasmid Group No.
Animal
the challenge infection nearly all of the swine of the E2-CD154 group reached this titer one week later.
3.3. Temperature and clinical signs after challenge
For a further comparison of the efficacy of the DNA-vaccine constructs and for a more detailed characterization of the influ-ence of the immunomodulating molecules, a measurement of body temperature and the determination of the magnitude of the CSFV-caused B-cell depletion after chal-lenge infection were included in the analy-ses. The body temperature of each group was measured daily after challenge. As expected, all pigs of the pcDNA4HisMAX-vaccinated control group 1 developed a high increase in body temperature four days after infection as well as typical CSFV-caused clinical signs and were euthanized on days 6–7 after challenge infection (Fig 4a). Three of four pigs that were immunized with the pcDNA4HisMAX-E2 plasmid (group 2) did not show any increase in their body temperature above 40 °C only one pig from this group had a temperature of 41 °C for one day (Fig. 4b). It is noteworthy that the individual with the highest temperature in this group was the animal with the lowest antibody titer presented in Figure 1, so that the low antibody might explain the increase in the body temperature based on an incom-plete protection. Interestingly, all pigs were free of other clinical signs. The immuniza-tion protocol for group 3, the group treated with the E2-IL-12 vector, failed in the pro-tection assay. Two pigs out of the four indi-viduals in group 3 who received the pcDNA4HisMAX-E2-IL-12 plasmid devel-oped fever over 40 °C which was accompa-nied by clinical signs, since there was dullness, weakness, anorexia and cyanosis of the skin. The affected animals had to be euthanized on days 6 and 7 after challenge infection (Fig. 4c). The other two individ-uals of this group did not show any increase of their body temperatures over 40 °C and were without pathological findings.
The other groups of animals in the E2-IL-18 and the E2-CD154 groups, which showed after vaccination in the NPLA a dis-tinct antibody titer (Tab. I), were all pro-tected after the challenge infection. Three out of four of the pcDNA4HisMax-E2-IL-18 plasmid immunized pigs (group 4) showed, during the experiment, a body tem-perature which did not rise above 40 °C; only one pig showed an increase in its body temperature to 41 °C for one day. All pigs were free of any clinical signs (Fig. 4d). A similar behavior was observed for group 5, the group of pigs that was immunized with the pcDNA4HisMax-E2-CD154 plasmid (Fig. 4e). Two pigs did not develop fever over 40 °C. The other individuals of that group showed temperatures above 40 °C for one and two days respectively. Also in that group, all pigs were free of any other clinical signs.
3.4. Depletion of CD21-positive B-lymphocytes after challenge infection
showed a reduction after four days post infection from 11% to 1%. This low per-centage of CD21+ B lymphocytes stayed
stable for several days until the animal had to be euthanized on day 10 p.i. The second
individual showed a decrease from 8% to 3% after four days p.i. and had to be sacri-ficed on day 7 p.i. The single member of the E2-vaccinated control group (group 2), which was analyzed during the course of the Figure 4. Body temperatures for all pigs were measured rectally every day after challenge infection.
infection, indicated a decrease of the rela-tive percentage of CD21-posirela-tive cells from 13% prior to the infection to 6% on day 4. Thereafter a low increase to 8% was detected. This animal died under sample collection on day 7 p.i. Unfortunately, only this single animal from the heterogeneous E2-group was analyzed, therefore we can only speculate about the fate of the B lym-phocytes of the other individuals of this group. Group three which was vaccinated with pcDNA4HisMax-E2-IL-12, the IL-12 containing construct, showed a heterogene-ous behavior. Two of the four animals dem-onstrated a severe depletion of CD21+ B
lymphocytes (9% to 2% and 18% to 1%, respectively) and had to be sacrificed on day 6 as well as on day 7 p.i. This behavior of the animals correlated with the body
tem-perature presented in Figure 2. The other two individuals of the group also showed a reduction at the onset of the infection (5% to 2% and 5% to 3%, respectively) but after 7 days p.i. both animals recovered and the percentage of B-lymphocytes stayed stable during the following days.
the absolute leukocyte numbers (data not shown).
3.5. Virus re-isolation after challenge
Besides the prevention of any clinical signs, a further criterion for the efficacy of a vaccine is its ability to avoid virus repli-cation in the respective tissues and virus spread to neighbor animals. For a quantifi-cation of the efficacy, virus titers were deter-mined in the blood, organs (kidney, spleen, mesenteric lymph node, liver, lung, tonsils) and nasal swabs by re-isolation.
As expected, all control animals (group 1) developed viraemia in the blood (> 5 × 103 infections virus particles/mL),
organs (> 5 × 103 virus particles/g) and nasal mucosa (> 1 × 103 virus particles/mL
nasal discharge) (Tab. III).
In group 2 vaccinated with the pcDNA4-HisMAX-E2 plasmid, none of the pigs showed any virus in the blood, organs and nasal swabs (Tab. III). This result was different from the result of the pcDNA4HisMAX-E2-IL-12 plasmid vaccinated (group 3). Besides the heterogeneities in clinical signs and B-cell depletion, this group was also heterogeneous with regards to viraemia. Virus could be isolated out of the blood from one pig (> 5 × 103 virus particles/mL)
and in tissue samples from another pig (kid-ney, spleen, mesenteric lymph node, liver, lung, tonsils > 5 × 103 virus particle/g). No
Table II. Percentage of CD21-positive B-lymphocytes in PBMC of pigs after CSFV challenge
infec-tion. The cross indicates euthanization of severely affected pigs. In groups 1 and 2 only several ani-mals were tested. The percentage of CD21-positve B lymphocytes was determined as described in the materials and methods.
Plasmid Group Animal No. dpi 0 dpi 4 dpi 7 dpi 10 dpi 14 dpi 20
virus was detectable in the blood, organs and nasal swabs derived from the two other members of this group (Tab. III). These data clearly correlated with the other clinical parameters and the survival of the individ-ual members of this group.
In group 4, vaccinated with the pcDNA4-HisMAX-E2-IL18 plasmid, as well as in group 5, treated with the pcDNA4HisMAX-E2-CD40L plasmid, CSFV could not be re-isolated in the blood, organs or nasal swabs of any of the individuals (Tab. III).
4. DISCUSSION
The glycoprotein E2 is the most immu-nogenic protein of the classical swine fever virus [6, 13]. It has been previously described that vaccination with the E2 glyco-protein plasmid DNA alone and in a prime-boost protocol with a recombinant porcine E2 adenovirus could protect pigs from lethal CSFV infection [18]. However vac-cination with an E2 marker vaccine does not inhibit horizontal and vertical virus spread [12].
Table III. Summary of viraemia in blood, different organs, nasal swab samples and survival from pigs
after CSFV infection. Positive tested piglets/ total no. of animals.
Plasmid
Animal No. Survival
Viraemia /TCID50/mL
In general cytokines play a primary role in the induction and regulation of immune response against infections [23] and as inte-grated parts of DNA vaccines, they are able to direct the antigen-specific immune response [20, 25, 43]. In this study we were able to show the effect of three immuno-modula-tory molecules in vitro. The NK activity was increased by using the pcDNA4-E2-IL-12 and pcDNA4-E2-IL-18 plasmids in vitro compared to the pcDNA4-E2 plasmid. The stimulating effect of pcDNA-E2 might be explained due to the CpG motifs in the plas-mid backbone [21]. Together with the results from the animal vaccinations, an influenceof the cytokines IL-12 and IL-18 and the co-stimulatory molecule CD154 on the immune response against CSFV was clearly demonstrated. For all constructs used for immunization with the exception of the pcDNA4HisMAX and the pcDNA4-His-MAX-E2-IL-12 expression plasmids, all pigs developed neutralizing antibodies as also described in other pre- and post challenge infections [42, 44].
In detail, vaccination with pcDNA4-HisMAX-E2-IL-12 only induced in one animal a low E2 specific neutralizing anti-body titer pre-challenge infection. After challenge, two of four pigs showed a prom-inent correlation between the lack of CSFV neutralizing antibodies, the development of fever and viraemia as well as the presence of CSFV in different organs. The one ani-mal who had developed CSFV neutralizing antibodies survived the challenge infection. In addition, the fourth animal who had not developed neutralizing antibodies also sur-vived similar to the described phenomenon after live vaccination [9]. Although T lym-phocytes seem to be involved in the devel-opment of a protective immune response against CSFV [2, 31] the presence of even low titer neutralizing antibodies is a good prognostic marker [17, 33]. Discussing the Th1/Th2 paradigma described for other species as for swine, our results suggest that the expression of IL-12 in conjunction with the E2 glycoprotein prevented the occur-rence of CSFV neutralizing serum antibodies
[37, 38] probably by a predominance of cel-lular response, e.g. of IFN- producing cells [52].
Vaccination with the pcDNA4HisMAX-E2-IL-18 and pcDNA4HisMAX-E2-CD40L plasmid was characterized in our experi-ments by the induction of high CSFV neu-tralizing antibody titers which were clearly linked to the lack of any clinical signs and the typical fever response as seen in the con-trol animals which all succumbed to the CSFV infection. Sporadic temporary increases of physiological body temperature after CSFV challenge infection was observed in this experiment but also occur after vacci-nation with E2-DNA vaccine [1] and after vaccination using a recombinant E2-Aden-ovirus [17].
Furthermore, all vaccinated pigs in group 4 (pcDNA4HisMAX-E2-IL-18) and group 5 (pcDNA4HisMAX-E2-CD154) had no virus detectable in the blood and organ samples. In the end, all animals of the two groups survived the lethal challenge infection, whereas one pig of the E2-control group with a lower neutralizing antibody titer had to be sacrificed due to severe clin-ical signs.
An additional important feature of viru-lent CSFV infection is the dramatic deple-tion of B lymphocytes [26, 39]. In contrast to the animals in other groups, we could detect in the blood of members of groups 4 and 5 vaccinated with pcDNA4HisMAX-E2-IL-18 and pcDNA4HisMAX-E2-CD154 plasmid DNA, a prevention of a long term B-lymphocyte decline after CSFV chal-lenge infection. A prevention of severe peripheral B-lymphocyte depression can also be explained by the stimulation of the protective humoral immune response as a function of IL-18 [50, 51] and CD40L [3, 16] co-expression in contrast to E2 alone and co-expression with IL-12.
Therefore excretion of the virus was determined by analyzing nasal swabs. There was no virus detectable in all groups with the exception of the non vaccinated control group (group 1). In our study, virus shedding could be inhibited by all of the DNA constructs used, even by the E2-con-struct alone. Our data on the suppression of virus excretion by the application of E2 alone are different from the results described by Dewulf et al. [12] who could show that application of E2 alone had no effect on virus spread after a challenge infection. These differences might be explained by the diverse application of E2. Dewulf et al. used recom-binant baculovirus-expressed E2 for immu-nization; we used an E2-DNA plasmid.
In contrast to the results from other stud-ies which failed to induce a protective immunity against CSFV by vaccinating with E2-DNA [1] and recombinant E2-con-taining adenovirus alone [18], we were able to induce a solid protection against virae-mia with the pcDNA4HisMAX-E2-IL-18 and pcDNA4HisMAX-E2-CD40L plasmid. This indeed shows the positive influence of IL-18 and CD154 on immunization with E2, but compared to the data from Andrew et al. [1] this effect could also be caused by our vaccination strategy by immunizing three times before challenge infection.
In conclusion, we can assume that DNA-vaccination with E2-IL-12 tends to a stim-ulation of a Th1 immune response and therefore to a suppression of an antigen spe-cific Th2-type immune response. In con-trast, vaccination with IL18 and E2-CD40L plasmids which seems to induce a Th2-type immune response protected pigs from a CSFV challenge infection, inhibited the nasal secretion of virus and enhanced the CSFV specific immune response com-pared to pigs vaccinated with an E2-plasmid alone. These results suggest that co-expres-sion of specific cytokines and co-stimula-tory molecules in combination with the E2 glycoprotein can enhance the efficacy of DNA vaccination against CSFV. The pro-tective potency of a single vaccination with
a cytokine/E2 combination will be the sub-ject of future investigations.
Taking these data together, we show that vaccination with an IL-18- or CD154- mod-ified E2-containing DNA plasmid led to the improvement of the immune reaction against a CSFV challenge infection. Interestingly, a combination of E2-DNA with IL-12 seemed to reduce the efficacy of the vaccine.
ACKNOWLEDGEMENTS
We thank U. Csacsko for technical support and W. Kramer for photo documentation. REFERENCES
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